Mitochondria occupy a central position in the overall metabolism of eukaryotic cells; hence the oxidative phosphorylation (OXPHOS), the Krebs's cycle, the urea cycle, the heme biosynthesis and the fatty acid oxidation take place within the organelle. Recently, another major role for mitochondria in determining the cellular life span was established, as they are recognized to be a major early mediator in the apoptotic cascade. Mitochondria are also a major producer of reactive oxygen species (ROS) causing oxidative stress and therefore inducers of cell death.
Primary defects in mitochondrial function are implicated in over 120 diseases and the list continues to grow, they encompass an extraordinary assemblage of clinical problems, commonly involving tissues that have high energy requirements, such as retina, heart, muscle, kidney, pancreas and liver. Their incidence is estimated of 1 in 5,000 live births. Indeed, combining epidemiological data on childhood and adult mitochondrial diseases suggests this prevalence as minimum, and could be much higher. Therefore, mitochondrial pathologies are considered among the most common genetically determined diseases, and are a major health issue since they remain inaccessible to both curative and palliative therapies.
Mitochondrion is assembled with proteins encoded by genes distributed between mitochondrial and nuclear genomes. These genes include those encoding the structural proteins of the respiratory chain complexes I-V, their associated substrates and products, the proteins necessary for mitochondrial biogenesis, the apparatus to import cytoplasmically synthesized precursors and the proteins necessary for mitochondrial assembly and turnover. Studies leading to the identification of genes involved in mitochondrial disorders have made considerable progress in the last decade. Indeed, numerous mutations in both mitochondrial DNA and a number of nuclear genes have been reported in association with a striking diversity of clinical presentations.
Approximately half of human mitochondrial disorders are caused by pathogenic point mutations of mtDNA, one-third of which are located in coding genes. There is currently no treatment for any of these disorders, a possible therapeutic approach is to introduce in the nucleus a wild-type copy of the gene mutated in the mitochondrial genome and import normal copies of the gene product into mitochondria from the cytosol. This approach has been termed “allotopic expression”.
There have already some reports describing that engineered nucleus-localized version of some mtDNA genes could be expressed in mammalian cells. For example, in a Leigh's disease case, a plasmid was constructed in which the mitochondrial targeting signal of the nuclear encoded COX8 gene was appended to a recoded mitochondrial ATP6 gene, mutated in patients. Stably transfected cells from patients present an improvement of growth in galactose medium and a mild increase in ATP synthesis, however the amount of Atp6 protein imported into mitochondria was relatively low (18.5%), implying that the precursor was not imported efficiently (Manfredi, G., et al., Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene to the nucleus. Nature Genet., 2002. 30: p. 394-399). Oco-Cassio and co-workers have demonstrated that allotopic expression of apocytochrome b and ND4 into Cos-7 and HeLa cells, did not lead to an efficient mitochondrial import of these proteins (Oca-Cossio, J., et al., Limitations of allotopic expression of mitochondrial genes in mammalian cells. Genetics, 2003.165: p. 707-720).
Hence, up today important limitations are found to the allotopic expression as a therapeutic approach and require optimization to overcome the significant hurdles before it can be applied in genetic therapy.
One hypothesis that can explain the poor import ability of the mitochondrial protein is its high hydrophobicity. Thus, the precursor synthesized in the cytoplasm remains stuck on the outer mitochondrial membrane.
Mitochondria assembly depends on balanced synthesis of 13 proteins encoded by mtDNA with more than a thousand others encoded by nuclear DNA. As the vast majority of mitochondrial polypeptides are synthesized in the cytoplasm, there is the requirement for an efficient and specific protein targeting system. This process involves the transport of mRNAs from the nucleus to the surface of mitochondria.
The inventors examined the possibility that allotopic expression of DNA such as mtDNA could be optimized by a targeted localization of the mRNA to the mitochondrial surface.